phase collapse
Posted: Mon Jul 19, 2010 10:26 am
Hi, I am not a very experienced chromatographer and suffering from the following problem:
I have used a column with a 100% aqueous mobile phase. I used a C18 column that apparently is not suitable for 100% aqueous mobile phases. The gradient used ran from 100% aqueous to 45% acetonitrile. At the end of the run I also shortly flushed with 100% acetonitrile before going back to the 100% aqueous mobile phase which started my gradient. The more samples I ran using this protocol, the worse the chromatography: severe peak broadening. I assume this is a result of phase collapse. I do, however, not see the shortened retention times associated with "phase collapse" (or de-wetting).
After finding out that I got such bad chromatography, I tried to regenerate the columns by flushing the column subsequently with methanol->isopropanol->hexane->isopropanol->methanol->80%acetonitrile/20%H2O. The chromatography stayed lousy, however, and I never got back the nice sharp peaks I used to get on this column. What might be the reason for this? Is it possible to regenerate a column after using a mobile phase containing 100% water?
thanks for your input,
Koen
I have used a column with a 100% aqueous mobile phase. I used a C18 column that apparently is not suitable for 100% aqueous mobile phases. The gradient used ran from 100% aqueous to 45% acetonitrile. At the end of the run I also shortly flushed with 100% acetonitrile before going back to the 100% aqueous mobile phase which started my gradient. The more samples I ran using this protocol, the worse the chromatography: severe peak broadening. I assume this is a result of phase collapse. I do, however, not see the shortened retention times associated with "phase collapse" (or de-wetting).
After finding out that I got such bad chromatography, I tried to regenerate the columns by flushing the column subsequently with methanol->isopropanol->hexane->isopropanol->methanol->80%acetonitrile/20%H2O. The chromatography stayed lousy, however, and I never got back the nice sharp peaks I used to get on this column. What might be the reason for this? Is it possible to regenerate a column after using a mobile phase containing 100% water?
thanks for your input,
Koen